BACKGROUND: Calcification is a natural process of bone formation or osteogenesis. However, calcium is able to be deposited abnormally in soft tissues such as the aorta, adipose tissue and liver, causing these to harden. Abnormal calcification in arteries is a common factor contributing to high blood pressure and, further, many severe cardiovascular diseases such as atherosclerosis and coronary disease. In liver and adipose tissue, calcification always takes place accompanied by excess extracellular matrix (ECM) accumulation which is called fibrosis, contributing to cirrhosis and metabolic disorders including insulin resistance. In addition, it is documented that severe calcification in adipose tissues is able to cause damage to the micro-vascular system, and calcification in perivascular adipose tissue (PVAT) is a key effector of arterial stiffness. Dystrophic calcification, one of the most common types of abnormal calcification, usually occurs as a reaction to tissue damage such as obesity-induced inflammation. Increasing numbers of studies indicate that abnormal calcification is the result of re-differentiation towards osteogenesis which occurs in the nascent resident cells under the stimulation of multiple factors. The BMP/Smad signaling pathway is commonly known to participate in bone formation and is implicated in mineralization as well as local induction of inflammation. Importantly, BMP/Smad signaling as an inducer of the osteochondrogenic phenotype in vascular calcification is fully appreciated. However, the molecular events of dystrophic calcification triggered by obesity-induced chronic inflammation still remain unclear. Our previous studies have identified that imbalance with increased activity of neutrophil elastase (NE), a Ser protease mainly released by neutrophils during inflammation, and decreased serum levels of the NE inhibitor α1-antitrypsin A1AT, contributes to the development of obesity-related metabolic complications including insulin resistance, fatty liver and chronic inflammation. This study explored the effects of NE on abnormal calcification in soft tissues, which may be mediated by BMP/Smad signaling pathway, and, furthermore, the molecular mechanism by which NE activates the BMP/Smad signaling pathway. METHODS: Wild-type mice were fed with either a high-fat high-fructose diet (HFHFD), a high-fat diet (HFD) alone or a normal chow diet (NCD), and NE-knockdown mice were fed with a HFHFD. Adipose tissue and liver were extracted from all mice. H&E staining and immunofluorescence staining (IF) detected the inflammation condition. Alizarin staining and von kossa staining were used to detect calcium deposits. 3,3′-Diaminobenzidine (DAB) staining was used to examine active phospho-Smad1/5 signaling. Regarding nascent resident cells which have potential ability of osteogenic re-differentiation, 3t3l1 fibroblast and human hepatic stellate cell (hHSC) were cultured in dishes and 6-well plates with coverslips. In our previous research, mouse aortic smooth muscle cells (mASMC) seeded in 6-well plates grew in an osteogenic medium (10mM β-glycerophosphate and 10mM Calcium chloride) in the presence or absence of NE (10nM). Calcium deposits were detected by Alizarin staining. 3t3l1 and hHSC was treated with NE (20nM, 30nM, 40nM), BMP2, TGFβ1 or NE combined with BMP2, TGFβ1 or NE inhibitor GW311616A (Axon). Further, we used specific chemical inhibitors, LDN-193189, BMP-ALK2/3 inhibitor, SB525334, TGFβ-ALK5 inhibitor, and I-191, PAR2 antagonist to investigate the molecular mechanism of NE’s effects on Smad signaling pathways. Cells in dishes were harvested, and the proteins were measured by western blot. Coverslips in 6-well plates were used for immunofluorescence. RESULTS: The most severe calcification was found in the adipose tissue of HFHFD fed wild-type mice and moderate calcification took place in the HFD mouse group while NCD mice rarely had calcium deposits. NE-knockdown significantly prevented calcium deposits in adipose tissue compared with HFHFD wild-type mice. Consistently, we found increased phospho-Smad1/5 (p-Smad1/5) signaling in the adipose tissues of mice on the HFHFD and HFD mice while p-Smad1/5 was prevented in the NE-knockout group. Furthermore, NE enhanced calcium deposits in mASMC cultured in osteogenic medium. NE significantly activated p-Smad1/5 signaling in hHSC in the dose-effect relationship and contributes to an additive effect on p-Smad1/5 in the presence of BMP2 or TGFβ1. Although p-Smad1/5 was only slightly aroused by NE in 3t3l1 fibroblast, NE was able to promote p-Smad1/5 activation tremendously and specifically in the presence of BMP2 or TGFβ1 but not p-Smad2/3 which is the main downstream signaling of TGFβ1. Chemical inhibition of ALK2/3, not ALK5 or PAR2, was able to completely block NE’s effects in hSHC on p-Smad1/5 activation. In addition, the cleavage of osteoblast-cadherin or CDH11 (OB-cadherin) was observed in hHSC, which may indicate a lower beta-catenin abundance in the hHSC cells which were treated with NE. CONCLUSION: NE has the potential to contribute to abnormal calcification in soft tissues including the liver, adipose tissue and aorta via activating canonical ALK2/3-BMP-Smad1/5 signaling pathway in the mesenchymal stem cell/MSC-lineage cells. In addition, NE is likely to break cell-cell adhesion which may contribute to cell proliferation and re-differentiation towards osteogenesis and fibrosis. / 2024-01-28T00:00:00Z

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19396号 / 医博第4047号 / 新制||医||1012(附属図書館) / 32421 / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 野田 亮, 教授 萩原 正敏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

In der vorliegenden Arbeit konnten erstmals der TGF-β-Signalweg und dessen Smad- Signalmoleküle innerhalb der MPCs nachgewiesen werden. Dieser Nachweis erfolgte sowohl auf zellulärer und Gewebeebene als auch auf Gen- und Proteinebene. Zusätzlich konnte auf Gen- und Proteinebene gezeigt werden, dass die Signalmoleküle Smad2, Smad3 und Smad4 in MPCs aus gering erkranktem Meniskusgewebe eine vermehrte Expression aufweisen im Vergleich zu den MPCs aus hochgradig erkranktem Meniskusgewebe. Diese Erkenntnis weist auf eine mögliche protektive Funktion des TGF- β-Signalwegs während degenerativer Prozesse im Meniskusgewebe hin. Um die Effekte des TGF-β-Signalwegs und dessen Smad-Signalmoleküle genauer zu verstehen und besser beschreiben zu können, wurde eine Überexpression der Smad-Signalmoleküle innerhalb von MPCs durchgeführt und die Auswirkungen dieser auf die Kollagen I- und Kollagen II-Synthese genauer beleuchtet. Infolgedessen konnte sowohl eine vermehrte Kollagen I-Synthese als auch eine vermehrte Kollagen II-Synthese festgestellt werden. Dies bestätigt die Annahme, dass dem TGF-β-Signalweg und dessen Smad-Signalmolekülen eine zentrale, protektive Funktion während der Meniskusdegeneration zukommt. Durch die vermehrte Synthese von Matrixkomponenten wird den Degenerationsprozessen innerhalb des Meniskusgewebes entgegengewirkt. Ein nicht degenerierter bzw. ein regenerierter Meniskus besitzt eine biomechanische Schutzfunktion für das Kniegelenk und wirkt somit einer Kniegelenkarthrose entgegen. In Zukunft könnte der TGF-β-Signalweg  einen möglichen Ansatzpunkt für therapeutische Behandlungen bei Meniskusläsionen darstellen. Da Meniskusdefekte häufig direkt mit einer Osteoarthrose im Kniegelenk assoziiert sind, spielt die durch den TGF-β-Signalweg induzierte Regeneration von Meniskusgewebe auch in der Prävention der Osteoarthrose eine zentrale Rolle.

Activin-A is a member of transforming growth factor-β (TGF-β) superfamily of cytokines which includes TGF-βs, Activins, Nodal, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs) and anti-Mullerian hormone (AMH). TGF-β, Activin and Nodal are known to activate SMAD2/3, while BMPs and GDFs are known to activate SMAD1/5/8 signaling pathways. Activin-A binds to type II transmembrane serine threonine kinase receptor (ActRIIA or ActRIIB), which in turn activates type I receptor (ActRIB) leading to phosphorylation of SMAD2/SMAD3. Upon phosphorylation, SMAD2/3 forms a complex with SMAD4, which then translocates to nucleus. In the nucleus, SMAD2/3/4 complex, along with other co-factors regulates expression of a large number of genes. Unlike TGF-β, role of Activin in cancer is not well understood. Activin has been shown to be overexpressed in several cancers including metastatic prostate cancer, colorectal cancer, lung cancer, hepatocellular carcinoma and pancreatic cancer. Activin signaling has been shown to promote aggressiveness of esophageal squamous cell carcinoma and enhancing skin tumorigenesis and progression. Nodal, which binds to the same set of receptors, has also been shown to be overexpressed in several cancers. However, role of Activins in breast cancer progression is not well studied. Activin is expressed by normal breast epithelium and is known to play a role in mammary gland development. Earlier, a study had reported downregulation of Activin signaling in breast tumors. On the contrary, increased serum level of Activin has been reported in women with metastatic breast cancers. It is pertinent to mention here that TGF-β, which has been implicated in the progression and metastatic spread of breast cancers, also functions through the same set of downstream effectors- SMAD2 and SMAD3. Hence we wanted to evaluate the status of Activin signaling pathway in breast tumors and investigate its functional role in cancer progression. Gene expression profiling of 80 breast tumors and 20 normal samples was earlier performed in our laboratory revealed overexpression of INHBA in tumors compared to normal tissue samples. An independent set of 30 tumor and 15 normal samples were used to verify these results. Real-time PCR analysis revealed around 11.31 fold upregulation (p<0.001) of INHBA in breast tumors in comparison to normals. While no change in expression of INHA was observed, INHBB was found to be significantly downregulated in tumor samples. These results indicated upregulation of Activin-A in breast tumors. Further, a significant upregulation of ACVR2A and SMAD2 which act as signal transducers of Activin signaling pathway, was observed in breast tumors. Interestingly, while an increase in the expression of TGF-β1 was observed, TGFBR2 was found to be significantly downregulated in breast tumors. In addition, PCR analysis revealed significant downregulation of FST, β-glycan, IGSF1 and IGSF10, which act as negative regulators of Activin signaling pathway. Functional antagonism between TGF-β/Activin and BMP signaling pathway has been shown in both development and disease. Further analysis revealed that various BMPs including BMP2, BMP4 and BMP6 are downregulated in breast tumors compared to normal tissue samples. Various components and regulators of BMP signaling pathway were also found to be deregulated, indicating suppression of BMP signaling in breast tumors. To evaluate whether Activin signaling is active in breast tumor cells, immunohistochemistry with another set of 13 normal and 29 tumor samples was performed. Immunohistochemistry analysis revealed that most of the tumors have higher levels of Activin-A compared to normals tissues. Interestingly, no significant changes in expression of Activin-A was observed between normals and low grade tumors, suggesting that Activin-A may play an important role towards the late stages of the disease. In good correlation, breast tumors showed increased phospho SMAD2 and phospho SMAD3 levels compared to normal tissues. Also, in the same set of tumors, BMP2 staining showed a reduced expression pattern compared to normal tissues. Expression of inhibin in some normal and breast tumor samples revealed that most of the tumor samples have lower levels of inhibin compared to normal tissues. In order to understand the role of Activin-A in cancer progression, a panel of cell lines was selected. Treatment of cells with Activin-A resulted in activation of canonical SMAD as well as non-canonical Erk1/2 and PI3K signaling pathways. However, Activin-A treatment did not lead to activation of TAK1/p38 MAPK pathway. To begin with, it was important to evaluate effect of Activin-A on proliferation of various cell lines. Primarily, SMAD2/3 signaling pathway inhibits proliferation of normal epithelial cells, and hence, it is considered to have a tumor suppressive role. owever, this signaling pathway remains intact in most ( 98%) of the breast cancers. BrdU incorporation assay showed that Activin-A does not promote proliferation of cells under monolayer culture conditions. However, soft agar assay results showed that Activin signaling promotes anchorage independent growth of cancer cells. TGF-β is widely known as an inducer of epithelial mesenchymal transition (EMT). Also, EMT is considered to be a prerequisite for epithelial cells to undergo migration and invasion. During EMT, cells loose epithelial characteristics and acquire mesenchymal features along with cytoskeletal rearrangement. Treatment of cells with Activin-A resulted in downregulation of E-cadherin and upregulation of various mesenchymal markers. In addition, confocal microscopy imaging revealed a mesenchymal morphology of cells treated with Activin-A. Also, collagen gel contraction assay results indicated that Activin-A enhances the contractile property of HaCaT cells significantly. Cells undergone EMT are believed to acquire migratory and Invasive behaviour. In agreement with this, both scratch assay and trans-well migration assay showed that Activin-A enhances the migration of various cell lines. Further, Trans-well matrigel invasion assays were performed to assess how Activin affects invasion of various cancer cells. Matrigel invasion assay results showed that Activin-A enhances invasion of various cancer cell lines significantly. Also, RT-PCR, zymography and Luciferase assay results showed that Activin-A induces MMP2 expression. As described earlier, Activin-A activates both canonical as well as non canonical signaling pathways. In this direction, it was interesting to investigate the contribution of SMAD signaling pathway in pro-tumorigenic actions of Activin-A. Inhibiting SMAD3 activity either by its stable knockdown or by using a SMAD3 specific small molecule inhibitor revealed that Activin-A regulation of EMT markers is SMAD3 dependent. Further, it was observed that SMAD3 contributes significantly in mediating Activin-A induced migration and invasion. Hence, it is likely that SMADs may play an important role in breast tumor progression. Next, stable overexpression of Activin-A in MCF-7 or its knockdown in MDA-MB-231 and H460 cells was performed to assess the effect of Activin-A on the behaviour of these cells. BrdU assay indicated no change in proliferation of cells upon overexpression or knockdown of Activin-A. However, soft agar assay results showed that Activin-A expression affects anchorage independent growth of these cells. MCF-7 cells are generally considered to be less aggressive in their tumor forming ability. Activin-A overexpressing MCF7 cells and control cells were respectively injected into right and left flank of immunocompromised mice and followed till the tumors reached to a prominent size. Our results show that Activin-A overexpressing MCF-7 cells have better tumor forming ability in comparison to control cells. In contrast to MCF-7 cells, MDA-MB-231 cells are known to be aggressive in their tumorigenic potential. In order to understand the effect of Activin-A knockdown on the tumor forming ability in MDA-MB-231 cells, 0.5 million cells (optimal cell number generally used is 1-2 million) were injected subcutaneously in immunocompromised mice. The results showed that while control cells gave rise to a tumor in 7 out of 10 animals, Activin-A knockdown cells could form a tumor in only 3 out of 10 animals. Also, the tumors formed by control cells were significantly larger by weight as compared to tumors formed by knockdown cells. Further, immunohistochemistry showed that tumors formed by MCF-7 cells overexpressing Activin-A have higher Ki-67 percentage as compared to control tumors. One of the factors known to be important for tumor growth is VEGF, which leads to recruitment of blood vessels and hence providing nourishment to the tumor cells. Hence Activin-A regulation of VEGF expression was evaluated next. Activin-A treatment or its stable overexpression in MCF-7 cells resulted in increased VEGF expression in these cells. This was also confirmed by VEGF promoter activity assay. To assess if Activin-A can play a role in metastatic spread of cancer cells, tail vein injection of Activin-A overexpressing MCF-7 cells was performed in immunocompromised mice. Even though no significant difference was found in the number of nodules formed by control or Activin-A overexpressing cells, it was observed that Activin-A overexpressing cells formed much bigger nodules as compared to the control cells. This suggests that Activin-A may play an important part in the establishment of metastases from the disseminated cancer cells. Tumor forming ability of cancer cells and aggressiveness of various cancers has been associated with the presence of cells having stem-like phenotype. In this direction, CD44high and CD24low expression status was analysed upon overexpression and knockdown of Activin-A in MCF-7 and MDA-MB-231 cells respectively. FACS analysis of Activin-A overexpressing MCF-7 cells and Activin-A knockdown MDA-MB-231 cells shows that Activin-A expression leads to enrichment of breast cancer stem-like cells. In conclusion, this study highlights the importance of Activin-A signaling pathway in the progression of breast tumors. It is also important to note the role of SMAD signalling in the progression of breast cancers since these effectors are common between TGF-β, Activin and nodal factors, which have been shown to be involved in cancer progression in a context dependent manner.

Der TGFβ-Signalweg ist ein multifunktionales System, das zelluläre Prozesse reguliert, die von Proliferation und Migration bis zu Differenzierung und Zelltod reichen. Nach Ligandenbindung und Rezeptoraktivierung translozieren SMAD-Proteine zum Zellkern und induzieren die Expression zahlreicher Zielgene. Während viele Komponenten des TGFβ-Signalweges identifiziert wurden, verstehen wir noch nicht genau, wie die Aktivierung des Signalwegs in verschiedene zelluläre Antworten übersetzt wird. Da die zelluläre Antwort auf einen gegebenen Stimulus oft sogar in genetisch identischen Zellen variiert, konzentrierte ich mich auf die Messung der Signalwegaktivität auf der Einzelzellebene. Durch die Kombination fluoreszierender Reporterzelllinien mit Zeitraffer-Lebendzellmikroskopie und automatisierter Bildanalyse beobachtete ich die zytoplasmatische und nukleäre Translokation von SMADs mit hoher zeitlicher und räumlicher Auflösung in Hunderten einzelner Zellen. Unsere Experimente zeigten, dass die Signalwegaktivität in eine erste synchrone Phase der SMAD-Translokation, gefolgt von einer Adaption und einer zweiten Signalphase mit hoher Variabilität in Stärke und Dauer der nuklearen Akkumulation unterteilt werden kann. Darüber hinaus beobachtete ich, dass Zellen, die aufgrund ihrer dynamischen Eigenschaften in Subpopulationen gruppiert sind, unterschiedliche phänotypische Reaktionen zeigen. Ich war nun daran interessiert, die Netzwerkinteraktionen zu identifizieren, die diese Dynamiken formen und fokussierte mich auf den Crosstalk mit nicht-kanonischen Komponenten des TGFβ-Signalweges. Ich konnte zeigen, dass die Hemmung der MAP Kinasen p38 und ERK die zweite Signalphase spezifisch aufhebt. Diese dynamische Remodellierung führt zu Veränderungen in der Zielgenexpression und den Zellschicksalen. Dies wird zu einem tieferen Verständnis der molekularen Netzwerke führen, die die TGFβ-Signaltransduktion regulieren und Möglichkeiten eröffnen, es in erkrankten Zellen zu modulieren. / The TGFβ pathway is a multi-functional signaling system regulating cellular processes ranging from proliferation and migration to differentiation and cell death. Upon ligand binding and receptor activation, SMAD proteins translocate to the nucleus and induce expression of numerous target genes. While many components of the TGFβ pathway have been identified, we are still challenged to understand how pathway activation is translated into distinct cellular responses. As the cellular response to a given stimulus often varies even in genetically identical cells, I focused on measuring pathway activity on the single cell level. By combining fluorescent reporter cell lines with time-lapse live-cell microscopy and automated image analysis, I monitored the cytoplasmic to nuclear translocation of SMADs with high temporal and spatial resolution in hundreds of individual cells. Our experiments demonstrated that pathway activity can be divided into a first synchronous phase of SMAD translocation, followed by adaptation and a second signaling phase with high variability in the extent and duration of nuclear accumulation. Furthermore, I observed that cells clustered into subpopulations according to their dynamic features show different phenotypic responses. I was interested in identifying the network interactions that shape these dynamics and focus on crosstalk with non-canonical components of the TGFβ pathway. I could show that inhibition of the MAP kinases p38 and ERK specifically abrogates the second signaling phase. This dynamic remodeling led to changes in target gene expression and cell fate decisions. I explored the molecular mechanisms underlying this interaction of the canonical and non-canonical pathways. This will provide a deeper understanding of the molecular networks regulating TGFβ signaling and open opportunities to modulate it in diseased cells.